TRANSPLANTATION (A PILEGGI, SECTION EDITOR)

Anti-Inflammatory Strategies to Enhance IsletEngraftment and Survival

Antonio Citro & Elisa Cantarelli & Lorenzo Piemonti

Published online: 4 August 2013# Springer Science+Business Media New York 2013

Abstract Early innate inflammatory reaction strongly affectsislet engraftment and survival after intrahepatic transplanta-tion. This early immune response is triggered by ischemia-reperfusion injury and instant blood mediated inflammatoryreaction (IBMIR) occurring hours and days after islet infusion.Evidence in both mouse model and in human counterpartsuggest the involvement of coagulation, complement system,and proinflammatory chemokines/cytokines. Identificationand targeting of pathway(s), playing a role as “master regula-tor(s)” in post-transplant detrimental inflammatory events, isnow mandatory to improve islet transplantation success. Thisreview will focus on inflammatory pathway(s) differentiallymodulated by islet isolation and mainly associated with theearly post-transplant events. Moreover, we will take into ac-count anti-inflammatory strategies that have been tested at 2levels: on the graft, ex vivo, during islet culture (i.e., donor)and/or on the graft site, in vivo, early after islet infusion (i.e.,recipient).

Keywords Islet transplantation .Early innate immune events .

Chemokines . Danger signals . Polymorphonuclear cells .

Instant blood mediated inflammatory reaction .

Anti-inflammatory strategies . Islet engraftment . Survival

Introduction

Type 1 diabetes (T1D) is an autoimmune disease associatedwith a permanent destruction ofβ-cells in the pancreatic islets.Actually the primary treatment for T1D, together with regular

monitoring of blood glucose levels, is daily and multipleinsulin injections. Although they have improved the patients’life, severe hypoglycemic episodes may occur and chronicdiabetic complications (i.e., nephropathy, neuropathy, andretinopathy) develop during the years. Currently, in order torestore a physiological glycemic control, an available alterna-tive is islet transplantation.

Clinical islet transplantation consists of islet infusion intothe portal vein where they engraft in hepatic microenviron-ment to release insulin. Protection from hypoglycaemicevents, improvement in HbA1c levels, and stabilization orreversion of secondary diabetic complications are primarytargets being addressed presently in the clinical practice, asreported by the International Islet Transplant Registry (www.citregistry.org). Advances in islet transplantation researchhave led to remarkable improvements in clinical transplantoutcome: considering the periods 1999–2002 and 2007–2010,the insulin independence rate is respectively 51 % and 66 %1 year post infusion [1••].

It is worthwhile mentioning that, as recently described bymany investigations in this field, substantial evidence suggeststhat an instant bloodmediated inflammatory injury (IBMIR) islargely responsible for the early functional stunning or de-struction of islets and may amplify subsequent adaptive im-mune reactions. IBMIR is a thrombotic reaction occurringwhen islets are incubated in ABO-compatible blood, mainlytriggered by production and secretion of tissue factor (TF) inislet preparations [2]. It is a non-specific innate immuneresponse related to mechanics and site, and further amplifiedby the graft, both contributing to ischemia-reperfusion injury.TF and pro-inflammatory mediators expressed by the grafttrigger the activation of the coagulation and complementcascades, clot formation, and leucocytes infiltration into theislets [3]. The evidence of these inflammatory innate immunereactions influencing islet engraftment and survival is primar-ily derived from quantitative positron emission tomographyscan imagery of labeled islets in humans [4] and from non-

A. Citro (*) : E. Cantarelli : L. PiemontiBeta Cell Biology Unit, Diabetes Research Institute, San RaffaeleScientific Institute, Via Olgettina 60, 20132 Milan, Italye-mail: citro.antonio@hsr.it

A. CitroDepartment of Surgery, University of Pavia, Pavia, Italy

Curr Diab Rep (2013) 13:733–744DOI 10.1007/s11892-013-0401-0

invasive magnetic resonance imaging studies in the mousemodels [5]. It is estimated that 60–80 % of the transplantedislet mass is lost within hours to days in clinical intrahepaticislet infusion mainly because of IBMIR, thrombosis, andhepatic tissue ischemia associated with elevated blood liverenzymes. Moreover, both pre-existing and transplant-inducedadaptive immune responses are involved and play a major rolein islet loss [6].

The recognition of these problems has increased theefforts in the identification of pathway(s) playing a roleas “master regulator(s)” of post-transplant detrimental inflam-matory events to be modulated both ex vivo, during culture inislet preparations (i.e., donor) and during the early innateinflammatory events occurring in the hepatic microenvi-ronment strictly site-dependent (i.e., recipient). Early andspecific targeting these innate inflammatory immuneevents could represent the ideal step toward a betteroutcome and management of islet-transplanted patients.In fact, emerging clinical data indicate that the use ofperi-transplant anti-inflammatory treatment positively im-pact both short- and long-term graft function after islettransplantation. Moreover, improving islet engraftment andfunction could contribute to achieve single-donor islet trans-plantation providing an expanding patient base with T1Dwithpoor glycemic control [7•].

This review is manly focused on promising recently devel-oped anti-inflammatory approaches tested first in preclinicalanimal models and then translated in the clinical practice toimprove islet engraftment and survival after intra-liver in-fusion. We will consider the experience in molecular path-ways differentially modulated after islet isolation and main-ly associated with the early innate inflammatory eventsprincipally taking into account anti-inflammatory strategiesthat have been tested or need to be tested at 2 levels:(1) on the graft, ex vivo, during islet culture (i.e.,donor), or (2) on the graft site, in vivo, early after isletinfusion (i.e., recipient).

The Donor: “Danger Signals” and Chemokines Secretedby Cultured Islets Involved in Survival and Engraftment

The injury response of islets because of isolation andpurification procedure may have negative consequencesby activating proinflammatory detrimental events at thegraft site. In particular, the isolation procedure exposesthe islets to a number of stresses that may adversely affect boththeir survival and function. Interventional strategies targetingdifferent molecules and pathways have been explored and testedover the last years to (1) prevent islet death; (2) preserve isletfunction; and (3) strictly limit the activation and/or amplificationof post-transplant detrimental innate inflammatory events.Numerous studies over the past decade have deeply described

the presence of pro-inflammatory molecules (ie, CXCL8/IL8,CXCL6/GCP-2, CXCL2/Gro-β, CXCL1/Gro-α, CXCL5/ENA78, CCL2/MCP-1, CXCL12/SDF-1, CCL28/MEC) and“danger signals” (ie, HMGB1, TF) induced by islet isolation.

A very well-known “danger signal” highly present onthe surface of human islets is the TF, the main mediator ofplatelets aggregation and activation. This material, onceinfused into the portal vein, triggers the IBMIR activation.After initial generation of thrombin, by TF-expressing is-lets, thrombin-activated platelets start to bind the surface ofthe islets. Via the amplification loop involving factor XIand activated platelets [8], more thrombin is formed, gen-erating a fibrin capsule surrounding the islets. Evidenceexists that clinical outcome of islet transplantation is direct-ly related to the extent of TF expression [2, 9], underliningthe relevance for the development of anti-coagulation ther-apies to prevent and/or reduce IBMIR. Several strategieshave been investigated in order to inhibit TF function suchas monoclonal antibodies, inactivated FVIIa, small molec-ular inhibitors, and siRNA [2, 9]. In current clinical prac-tice, islets are transplanted in heparinized medium to pre-vent coagulation. Low-molecular-weight dextran sulphate(LMW-SD) has been suggested to replace heparin becauseit is able to block IBMIR to a greater extent in comparisonwith heparin in vitro [10, 11] and significantly prolongssurvival of intraportally transplanted islets in vivo [12].Based on this evidence, a clinical trial to assess safetyand effectiveness of LMW-SD on post-transplant islet func-tion in people with T1D was planned (NCT00790439) butwithdrawn because of funding limitations; no results werefurther reported. In this context, islet surface heparinizationcould be an attractive alternative to soluble heparin, ren-dering islet surface biocompatible when exposed to theblood and to avoid the risk of systemic complications suchas bleeding. In addition to the effects on the coagulationcascade, heparin coating is able to reduce exposure ofcollagen and other extracellular matrix proteins on the isletsurface that may have a role in triggering thrombosis andinflammation [13]. Similar results overcoming IBMIR havebeen obtained by creating a composite islet-endothelial cellgraft co-culturing islets with primary aortic endothelial cells.This innovative approach have led to the reduction of coagu-lation and complement activation with decreased plateletsconsumption and leucocytes infiltration both in vitro andin vivo [14, 15]. In addition, many new anti-coagulant inhib-itors have been shown to prevent islet damage in vitro:melagatran, a specific thrombin inhibitor [16]; nacystelyn (aderivative of N-acetylcysteine), a blocker of TF mRNA trans-lation [17, 18]; and nicotinamide [19, 20]. Among them,nacystelyn is currently used in clinical practice in ischemia-reperfusion injury prevention after liver transplantation [21,22] and melagatran in anticoagulation therapy [23]. However,no clinical data exist in islet transplantation, mainly because all

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these strategies have shown only a modest benefit in a limitedseries of preclinical in vivo studies. The main reason could bethat it is unlikely that an agent targeting only one component ofIBMIR would block all elements of the reaction (i.e., coagu-lation, complement activation, production of proinflammatorymediators); therefore, more than one agent needs to be used toimprove islet engraftment.

Very recent studies suggested the relevance of the donor-derived high-mobility group box-1 protein (HMGB1) asanother “danger signal” involved in engraftment and sur-vival of intraportally infused islets. For the first time,Matsuoka et al. have clarified the key role of HMGB1 inearly innate inflammatory events crucial for islet engraft-ment and survival in the mouse model. Treatment with ananti-HMGB1 antibody prevented early graft loss byinhibiting IFNγ production by NKT cells and Gr1-

CD11b+ cells. Moreover, targeting other molecules in-volved in NKT cells activation and subsequent NKT cell-dependent IFNγ production by Gr1- CD11b+ cells such asIL-2 or CD40L, prevented early graft loss [24•]. Furtherstudies correlating HMGB1 secretion with graft function bytransplanting human islets into diabetic mice, have shownthat the amount of released HMGB1 reflected the degree ofislet damage and correlated with the negative outcome ofislet transplantation [25]. Last year, for the first time, Nanoet al. has reported that human islet preparations containedhigh levels of HMGB1 after isolation, mainly caused bytissue damage and necrosis rather than by NF-κB or TLRstimulation. Although HMGB1 levels were found to besignificantly positively associated with the release of thepro-inflammatory cytokines and chemokines CXCL8/IL-8,CXCL1/Gro-α, IFNγ, CXCL10/IP-10, and CXCL9/MIG,no correlation with short-term human islet function wasreported [26]. On the contrary, inverse correlation betweenin vitro HMGB1 levels and graft function has been de-scribed in the clinical practice in an autologous setting [27].

Numerous evidence suggested that “danger signals” arecommonly associated with pro-inflammatory molecules in-duced by NF-κB and TLR activation in leukocytes and fibro-blasts [28–31]. A very recent genome-wide transcriptionalstudy of human β-cells obtained by laser capture microdissec-tion from whole pancreas and islets freshly isolated (day 0) andafter 3 days culture (day 3) reported an upregulation ofproinflammatory molecules. In particular, the CXCR1/2 ligandCXCL8/IL-8, CXCL6/GCP-2, CXCL2/Gro-β, CXCL1/Gro-α, and CXCL5/ENA78 were the most upregulated uponisolation both at day 0 and day 3 [32••]. Similar results havebeen reported also by Cowley et al. demonstrating theupregulation of a panel of genes closed to the family ofchemokines and crucial for neutrophils and monocytes recruit-ment (CXCL2/Gro-β, CCL2/MCP-1, CXCL12/SDF-1,CXCL1/Gro-α, CXCL6/GCP-2, and CCL28/MEC) [33••]. Inthe same direction, we have described the upregulation of the

chemokines CXCL8/IL-8, CXCL1/Gro-α, and CCL2/MCP-1in human islet surnatant during the first 24 hours of culturebefore transplantation [34••]. Although the pro-inflammatorysignature of islets after isolation have been very well-describedand characterized, no specific interventional strategies of ex vivoislet pre-treatment targeting these pathway(s) was demonstratedto be efficient in improving transplant outcome. Probably themain reason could be the redundancy and promiscuity of thechemokine/chemokine receptor system and, thus, ex vivotargeting a single pro-inflammatory axis seems not to besufficient to achieve an improvement in islet engraftmentand function. However, numerous studies have correlatedpre-transplant pro-inflammatory molecule release as sign of“damaged” islets with post-transplant islet function. Amongthem, CCL2/MCP-1 has been largely investigated in themouse model. In fact CCL2−/− transgenic mice have beenused to demonstrate that donor CCL2 did not affect isletsurvival and function, whereas the inhibition of recipientCCL2 led to a significant improvement in graft functionwith partial abrogation of local hepatic inflammation bothin syngeneic [35] and allogeneic [36, 37] transplantationsettings. When evaluated in cultured human islets, CCL2release appeared strongly related to the immediate localinflammatory response in the liver and to short-term humanislet function [38]. Definitely, CCL2/MCP-1 release is a signof “inflamed” islets significantly and positively associatedwith other cytokines/chemokines, in particular with the high-ly released pro-inflammatory IL-6 and CXCL8/IL-8 orCXCL1/Gro-α, all impacting on islet engraftment.

In this scenario, potential anti-inflammatory moleculeshave been added to the islets in culture after isolation toprevent ex vivo secretion of pro-inflammatory molecules. Ofnote NF-kB, the main transcription factor regulating the ex-pression of several chemokines and cytokines (CCL2/MCP-1,CXCL10/IP10, and IL-15) that promote the migration ofinnate immune cells [39], has been considered a promisingtarget. In this context, evidence in the mouse model of im-proved islet engraftment after conditional NF-κB inhibition inβ-cells [40–42] has led to experimental islet pre-treatmenttargeting NF-κ through ex vivo gene therapy [43], curcumin[44], and dehydroxymethylepoxyquinomicin (DHMEQ) [45].A very recent study have explored a possible therapeuticoption using ex vivo pharmacologic pre-treatment with theproteasome inhibitor Bortezomib, able to inhibit NF-κ activa-tion in the islets finally reducing cellular stress response,apoptosis, and CXCL10/IP10 release [46•].

Ex vivo islet pre-treatment really represents a crucial stepfor potential tissue manipulation prior to implantation in orderto reduce or prevent in vivo IBMIR activation by decreasingthe early non-specific inflammation. Much effort to identifyex vivo master regulator(s) with an in vivo impact on trans-plant outcome have been carried forward (Fig. 1). The majoradvantage of ex vivo pre-treatment is the localized effect at

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graft site overcoming systemic side effects. However, it seemsthat multiple strategies need to be used to address the

numerous pathway(s) involved in the complex innate inflam-matory process early occurring after islet infusion.

Fig. 1 Evaluation of different “master regulator(s)” ex vivo (donor) andin vivo (recipient) targeted in islet transplantation. A, Dithizone-stainedmouse islets. B, Hematoxylin/Eosin mouse hepatic tissue 3 days afterallogeneic islet transplantation. I islet, L.I. leucocyte infiltration, N

necrosis. Green = tested in clinical trial; Orange = promising preclinicaldata, not yet translated in the clinical trial; Red = not promising preclinicaldata; n.t. = not targeted

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The Recipient:Master Regulator(s) Early Involved in IsletEngraftment and Survival After Intra-Liver Infusion

Pancreatic islet infusion directly into the portal vein causescells entrapment in the terminal venous liver branches. Theclosed proximity to the vascular space provides both nutri-tional and physical support for the islets which, after isolationand purification process, have lost their dense vasculature andspecialized extracellular matrix. On the other hand, the hepaticportal vasculature could represent an hostile hepatic microen-vironment due to low oxygen tension and poor revasculariza-tion compared with native islets in the pancreas [47, 48], highglucose levels [49], high level of immunosuppressive drugs[50] and local insulin-induced hepatic steatosis [51, 52] thatfinally limit islet engraftment and survival. All these phenom-ena severely reduce the total islet mass and represent the mainfactor necessitating a very large number of islets to achievenormoglycemia [53•]. For this reason, islet transplantationresearch really needs to focus on early nonspecific innateinflammatory events to increase the total islet mass survivingafter infusion and finally provide a significant challenge tosubstantive improvements.

Characterization of the pro-inflammatory status early (ie,hours and days) after intra-hepatic islet infusion resulted in theincrease of numerous chemokines, cytokines, and “dangersignals” in the peripheral blood. Among them, it is alreadywell-known that there is a significant elevation in circulatingliver enzymes (i.e., aspartate aminotransferase and alanineaminotransferase) occurring during the first week and normal-izing spontaneously within the first 4 weeks post-transplant.This elevation could be induced by multiple factors such ashepatocytes damage, hypoxic injury, and immunologic reac-tions toward newly infused islets [54, 55].

A pivotal mediator in ischemia-reperfusion injury and inthe pro-inflammatory reaction including chemotaxis, cell ac-tivation, and cytokine release is the complement system, assuggested by in vitro direct antibody-mediated complementactivation on the islet surface [56, 57] and by inhibition ofcomplement activation by thrombin inhibitors [16]. Notablyanaphylatoxins C3a and C5a, released upon activation ofIBMIR cascade events, are supposed to be responsible, atleast in part, for leukocyte recruitment, and infiltration intotransplanted islets. Different compounds targeting the com-plement system have been tested in vitro and/or in vivo:C5aIP, the C5a inhibitory peptide [58]; sCR1, and TP10,soluble complement receptor 1 inhibitors [59, 60].Collectively, complement 5a factor (C5a), through bindingto its receptor C5aR, mediated the exposure of TF on neutro-phils, thereby significantly enhance their pro-coagulant activity.Therefore, treatment with C5aIP has been shown to improvethe outcome of intraportal islet transplantation by attenuatingthe cross-talk between complement and coagulation cascadesboth by inhibiting C5a-C5aR binding and by suppressing TF

expression on granulocytes in the host livers with only amarginal effect on cytokine inhibition [58, 61]. A possiblemechanistic explanation could be derived by the evidence thatC5a/C5L2 (ie, a secondary receptor for C5a) interaction in-duced HMGB1 release in a mouse model of acute lung injury[62], even if few differences were observed in the circulatingHMGB1 levels (ie, marker of “damaged” islets) after isletinfusion in the presence or absence of C5aIP. Even thoughthese molecules have been largely studied in syngeneic andxenogeneic models of islet transplantation, they have not beentranslated into the clinical practice till now. In clinical ischemia-reperfusion injury treatment, different molecules targeting thecomplement system have been tested: C1 esterase inhibitor inemergency coronary artery bypass and capillary leakage syn-drome after cardiopulmonary bypass and sCR1 in cardiopul-monary bypass [63, 64]. However, in these clinical settings, nobeneficial effects have been observed probably because in vivoactivation of the complement system is diffuse and difficult todetect. Moreover, paradoxically, controlled complement activa-tion can also be protective against ischemia-reperfusion injuryand activate mechanism of tissue remodeling.

Numerous factors, other than complement system, elicitedIBMIR such as the coagulation cascade, targeted not onlyex vivo as previously mentioned, but also in vivo. In thiscontext, a study in a non-human primate marginal mass modelshowed that TF inhibition through a monoclonal antibody(CNTO859) could enhance islet engraftment (ie, lower levelsof post transplant markers of coagulation, higher fasting C-peptide levels at 1 month post-transplant), and prolonged graftfunction [65]. In this context, an innovative approach recentlydeveloped consists of islet coating with a thin polymer filmcontaining thrombomodulin (TM), a thrombin inhibitor [66,67]. Cui et al. have clearly demonstrated in a syngeneic mousemodel of intra-liver islet transplantation that TM treatmentsignificantly improved early islet engraftment affecting bothcoagulation (i.e., marked reduction in intra-portal fibrin for-mation) and inflammation (ie, marked reduction in neutrophilsinfiltration and IL-1β, TNF-α, IFNγ, NO) pathways [68].This innovative strategy creates a local anti-inflammatorymicroenvironment with limited graft-triggered coagulationcascade, inhibition of thrombin formation and reduction inplatelet activation, and leukocytes recruitment.

Reduction and/or inhibition of complement and/or coagu-lation cascade events have been demonstrated to limit massiveleukocyte recruitment at graft site [16] strongly elicited bypro-inflammatory mediators released by the islets. Actually,the mechanism(s) by which the newly transplanted isletsstimulated polymorphonuclear leukocytes (PMNs) and mono-cytes recruitment were not completely understood. PMNwerethe predominant cell type infiltrating the islets in vitro,appearing 15 minutes after incubation with ABO-compatibleblood with massive infiltration within 1 hour and peaked at2 hours [69]. In vivo evidence showed that PMN, mostly

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CXCR2+ cells, infiltrated the necrotic hepatic regions startingfrom day 2 after allo-transplantation [70]. Overall, the infil-tration pattern observed during the IBMIR resembled, at leastin part, that detected after ischemia-reperfusion injury: PMNwere attracted to the graft site due to pro-inflammatory medi-ators and chemotactic molecules released both by infusedislets (i.e., donor) and by hepatic microenvironment (i.e.,recipient).

Among them, CXCL8/IL-8 is the most relevant pro-infla-mmatory factor found both in transplanted patients and in themurine counterpart after experimental islet transplants. Our re-cent findings in themousemodel confirmed that CXCL1/Kc (ie,murine CXCL8 functional homologue) is strongly and signifi-cantly up-regulated early at graft site (i.e., liver) [34••].Moreover, CXCL8/IL-8 is highly expressed by the graft, bothupregulated in human isolated islets [32••] and secreted in thesupernatant of murine islets [35]. CXCL8/IL-8 was a crucialmediator of PMN recruitment into transplanted islets and thusreally represented, by binding to its receptors CXCR1/2, aprimary therapeutic target to control ischemia-reperfusion injuryand finally prevent early graft failure [71–75]. In fact in apreclinical model of intra-liver islet transplantation, earlytargeting CXCL1-CXCR2/1 axis genetically and pharmacolog-ically, resulted in improved islet engraftment and delayedgraft rejection respectively in syngeneic and allogeneic set-tings. Supporting evidences suggested that the massive PMNinfiltration could cause direct damage to the islets, not onlyby functionally impairing or reducing islet transplanted mass,but probably also amplifying the subsequent adaptive im-mune responses. On the basis of promising preclinical results,a phase 2 pilot study (NCT01220856) was conducted toassess the efficacy and safety of Reparixin (i.e., allostericnoncompetitive inhibitor of CXCR2/1) after a single isletinfusion in T1D patients and a phase 3, multicenter, random-ized, double-blind clinical trial is now ongoing [34••]. Forthe first time, both preclinical and clinical successfulresults support the relevance of early peri-transplantanti-inflammatory treatment targeting chemokine-driveninnate inflammatory events to positively impact bothshort- and long-term graft functions after islet transplan-tation [76].

It is well-known that other pro-inflammatory moleculessuch as TNF-α and IL-1β are released by isolated islets inresponse to stress (i.e., donor), by activated PMN and macro-phages recruited after ischemia-reperfusion injury and by Tcells during graft rejection (i.e., recipient) [77]. In this scenar-io, encouraging results were reported by targeting TNF-α [76,78, 79, 80] and IL-1β [81] axis.

It has been shown that TNF-α exposure is cytotoxic tohuman islets in culture [82] and has been proposed as crucialcontributor for early peri-transplant graft loss [83]. In a pre-clinical model of intra-hepatic islet transplantation, Bottinoet al. have suggested that high levels of pro-inflammatory

mediators such as TNF-α and IL1-β were associated withpoor graft function. Moreover, inhibition of macrophage acti-vation improved islet function by reducing the release of theseearly pro-inflammatory mediators. These results suggest thatreduction and/or inhibition of TNF-α and IL1-β release facil-itate the engraftment of allogeneic islets in the liver [84].Firstly in islet transplantation research, Farney et al. havedemonstrated in a syngeneic mouse model the potential ben-efit of TNF-α inhibition [85]. More recently, proof of princi-ple that in heart allograft mouse models, resembling ischemia-reperfusion injury, TNF-α inhibition substantially reducedearly intra-graft leukocyte infiltration and prolonged graftsurvival [86], supported the potential efficacy of moleculestargeting TNF-α axis in the prevention of early islet loss. Onthe basis of the single study by Farney et al., the application ofTNF-α inhibitor (Etanercept) has become integrated into theclinical practice for allogeneic islet transplantation, withoutpreclinical data on the efficacy of this drug. Several clinicalstudies reported insulin independence achievement after sin-gle donor marginal islet mass transplantation when peri-trans-plant TNF-α inhibitor was used in combination with theconventional immunosuppressive regimen including T celldepleting agents [78, 80, 87]: during the era 2007–2010 therate of insulin independence 3–5 years after last infusion was50–62 % and 34–43 % in recipients receiving or not receivingthis regimen respectively [1••]. These clinical successful resultsstrongly support the idea that long-term islet survival is highlydependent upon the initial inflammatory events that couldinfluence pre-existing and transplant-induced adaptive immuneresponses occurring at graft site. However, the use of a differentTNF-α inhibitor, such as Infliximab, did not reproduce a de-monstrable clinical benefit [88], probably due to lower dosagesand/or shorter duration of treatment to limit early innate inflam-matory events. Furthermore, the different clinical outcome couldbe explained by certain biological differences between the twoTNF-α inhibitors. Both Etanercept and Infliximab, by bindingthe free soluble TNF-α, prevented its binding to the TNF-αreceptor; however, only the IgG1 antibody molecule Infliximabcould activate complement or initiate antibody dependent cellu-lar cytotoxicity, thereby causing cytolysis of cells bearingTNF-α on their surface, such as activated macrophages [89].

Besides TNF-α, IL-1β is an early pro-inflammatory cyto-kine released by activated PMN and macrophages [61, 90]. Itis crucial in upregulating the expression of the genes for nitricoxide (NO), prostaglandins (PGs), reactive oxygen interme-diates (ROIs), and cyclooxygenase-2 (COX-2) further affect-ing islet survival by causing graft dysfunction and/or apopto-sis [91]. Preclinical studies have demonstrated that treatmentwith IL-1 receptor antagonist (IL-1Ra) clearly improved trans-plant outcome in islet transplantation under the kidney capsulein several transplant settings: syngeneic in NOD mice [92],allogeneic, and xenogeneic in alloxan-induced mice [93].Moreover, a preclinical study has demonstrated that treatment

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with IL-1Ra (Anakinra) significantly improved islet function byattenuating islet amyloid polypeptide-induced pro-inflammatorycytokine release [94]. Very recently, McCall et al. have shownthat the association of IL-1Ra and TNF-α inhibitors resulted inislet engraftment improvement in comparison with TNF-α in-hibitor alone in a marginal mass model of islet transplantationunder the kidney capsule [95•]. Data supporting the efficacyboth of IL-1Ra and TNF-α inhibitors have been largely obtainedin a mouse model of islet transplantation under kidney capsulethat does not reproduce ischemia-reperfusion injury and IBMIR,commonly induced by islet infusion into the portal vein [70].Although the lack of appropriate and robust supportive preclin-ical data, a clinical trial started to assess safety and tolerability ofIL-1Ra (Anakinra) and TNF-α inhibitor (Etanercept) in associ-ation with T-cell depleting agents. Although the treatment hasbeen considered safe, this report was limited to a small numberof patients at single institute; thus, multicenter investigation in alarge cohort of patients should be performed in order to evaluateefficacy [96•].

Finally, a variety of approaches have been explored toprevent the apoptotic destruction of the islets in the experi-mental setting and, while promising data have been generatedin vitro, evidence of in vivo efficacy has been more tricky.Such strategies included both approaches with gene therapy aswell as pharmacological interventions. Among them, ex vivoadenoviral-mediated gene transfer of the anti-apoptotic Bcl-2gene protected the islets from apoptosis and in vivo conferredlong-term, stable protection, and maintenance of functionalislet mass after transplantation of macaque islets into diabeticsevere combined immunodeficient mice [97]. Moreover,transduction with a XIAP-expressing recombinant adenovirusof human and mouse islets, a potent endogenous inhibitor ofthe downstream effector caspases 3, 7, and 9, showed verypromising results. This ex vivo gene therapy strategy was ableto induce resistance to apoptosis, functional recovery follow-ing in vitro hypoxia stress, and reduction in islet mass requiredto reverse hyperglycaemia in chemically-induced diabeticimmunodeficient mice [98, 99]. Other anti-apoptotic geneticstrategies comprised transduction with a chimeric adenovirusvector to enhance SOCS1 (suppressor of cytokine signaling 1)expression and the over-expression of the anti-apoptotic A20[100, 101]. Notably, while experimental data on genetic mod-ification of pancreatic islets seem to be promising to minimizeislet mass loss after transplantation and reduce the number ofislets to achieve normoglycaemia, actually, there is no trans-lation of the anti-apoptotic gene therapy strategies into clinicalpractice mainly because of the persistence of safety concernstill now. In the last decade, much effort has been focused onthe development of anti-apoptotic pharmacological agents.Among them, the caspase inhibitor zVAD-FMK, already usedin several animal models of ischemia-reperfusion injury, wasrecently tested in a syngeneic mouse model of islet transplantboth under kidney capsule and in the liver. This compound

have been preclinically used both ex vivo and in vivo dem-onstrating that ex vivo islet pre-treatment had only a marginalimpact on graft function [102], whereas both ex vivo andin vivo peri-transplant treatment improve long-term marginalmass islet function [103]. Similar results have been obtainedalso in a xenogeneic mouse model of human islet transplant[103, 104]. Another molecule targeting caspase, IDN-6556,already used in the clinical practice after liver transplantation,protected against ischemia-reperfusion mediated apoptosisand injury [105]. Recently McCall et al. have tested theefficacy of IDN-6556 in vivo treatment both in a syngeneicand xenogeneic marginal mass model of islet transplantationunder the kidney capsule. In both experimental settings lowernumbers of islets were required for normoglycaemia achieve-ment [95•, 106••]. Evidence of the efficacy and safety of IDN-6556 both in the large animal model [107] and in humans andthe ease route of administration (i.e., oral) clearly simplify afuture clinical translation in islet transplanted patients. Peri-transplant targeting apoptosis really represents a critical pointin controlling early innate inflammatory events.

In this context, another innovative approach consists intargeting glucagon-like peptide-1 (GLP-1) as anti-apoptoticstrategy to increase β-cells mass thus improving graft out-come. It is also well-known the effect of GLP-1R agonists inreduce the release of pro-inflammatory molecules (ie, TF,IFN- γ, IL-17, IL-1β, and IL-2) ex vivo by human islets[108]. Actually, there are 2 GLP-1 receptor agonists approvedfor the clinical practice: (1) Exendin-4 (also called Exenatide)with short half-life and (2) Liraglutide (also called Victoza)with long half-life. This approach have been tested both asex vivo treatment of mouse and human islet preparations andas in vivo treatment after islet infusion. Indeed, Weir et al.clearly demonstrated that ex vivo treatment of murine isletswith exendin-4 increase the rate of hyperglycaemia reversal,although in vivo treatment with exendin-4 for 14 days posttransplant did not exhibit any beneficial effect on glucosehomeostasis [109]. Moreover, Toyoda et al. reported a bene-ficial effect of GLP-1 in restoration of normoglycemia in aminimal intra-hepatic mouse model of islet transplant demon-strating a significant reduction in the number of apoptotic β-cells during early post-transplant phase [110]. A very recentstudy confirmed that ex vivo treatment of porcine and murineislets with exendin-4 improves the outcome of syngenic andxenogenic islet transplantation [111]. In addition, the Shapirogroup has evaluated the efficacy of GLP-1 targeting in a syn-geneic marginal mass model of islet transplantation and provedthat liraglutide treatment induce normoglycaemia achievementearlier after infusion in comparison with sirolimus by reducingβ-cell apoptosis [112]. However, liraglutide did not have anyadvantage on insulin independent rate in a marginal massporcine islet autografts [113]. In clinical islet transplantation,GLP-1 is used as a possible target in association with an anti-inflammatory therapy to (1) improve insulin secretion, (2)

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preserve islet mass, and (3) reduce apoptosis associated withearly innate inflammatory events. Very recent clinical trialshave evaluated the efficacy of exenatide in association withetanercept in islet transplanted recipients. Proof of principle ofbetter islet engraftment and long-term graft survival and func-tion in subjects undergoing supplemental islet infusion wasreported in a non-randomized and small sample size study byAlejandro group [79]. In addition, the Oberholzer group havedemonstrated that exenatide and etanercept in the presence ofEdmonton immunosuppression protocol required lower num-ber of islets for insulin independence achievement [87].

Taken together, preclinical and clinical data strongly rec-ommend the introduction of anti-inflammatory drugs (ie,targeting early innate inflammatory immune response) incombination with the traditional immunosuppressive therapy(i.e., targeting adaptive immune response). However, thetiming of anti-inflammatory molecules exposure is likely crit-ical and these strategies need to be administered for shortperiod of time (i.e., days) to avoid unwanted side effectsmainly due to the ubiquitous expression of these targets inseveral cell types and tissues. Moreover, in order to indentify“master regulator(s)” involved in early innate inflammatoryevents and to further select promising strategies that may betranslated into the clinical practice, the design of meticulouspreclinical studies represents a critical point to obtain consis-tent and reliable results. Indeed the routine peri-transplantclinical adoption of anti-inflammatory approaches such asEtanercept and/or Anakinra is generally empiric and oftenlacks appropriate and robust supportive preclinical [85]] orclinical data [78, 81]. In this context the choice of a suitablepreclinical model need to consider several experimental pa-rameters (i.e., mouse models of diabetes, islet number, site oftransplant, dose and administration of the drugs) that couldfurther influence islet transplant outcome. Another criticalpoint that needs to be considered in preclinical research is thatsome discrepancies both in innate and adaptive immune re-sponses really exist between the mouse model and men andneed to be taken into account in the adoption of mice as apreclinical model of the human counterpart [114].

Conclusions

Islet transplantation is a very dynamic field. In the past years, ithas becoming increasingly recognized that anti-inflammatorystrategies need to be introduced into the peri-transplantperiod to reduce and/or prevent the early innate inflamma-tory events that strictly affect islet survival and engraftmentin liver microenvironment. Numerous approaches ex vivoand/or in vivo targeting different “master regulator(s)” havebeen investigated: (1) coagulation cascade, (2) complementsystem, (3) pro-inflammatory mediators both secreted by thegraft and upregulated at graft site, (4) PMN recruitment, and

(5) apoptosis (Fig. 1). Both selectivity and timing of thetreatment are critical points to specifically address detrimentalinflammatory events both avoiding systemic side effects andpreventing tissue remodelling. Moreover, translational re-search, as the field of islet transplantation is, needs verycontrolled preclinical studies that try to closely mimic thehuman counterpart to have reliable results before clinicaltranslation. Clinical trials studying safety and efficacy ofpromising anti-inflammatory molecules targeting CXCR1/2,TNF-α, IL-1β, and caspase are ongoing. Clinical preliminaryresults and longer follow-up will be needed to fully demon-strate the benefit on transplant outcome.

Compliance with Ethics Guidelines

Conflict of Interest Antonio Citro declares that he has no conflict ofinterest. Elisa Cantarelli declares that she has no conflict of interest.Lorenzo Piemonti has received grant support from Dompe S.P:A. forsupport with a support pre-clinical study with CXCR1/2 inhibitor andclinical Trial NCT01220856.

Human and Animal Rights and Informed Consent This article doesnot contain any studies with human or animal subjects performed by anyof the authors.

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